Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain

Hydrophilic insulins, more positively charged than human insulin at neutral pH, have been prepared by substitution with basic amino acids at the termini of the B-chain and by blocking the C-terminal carboxyl group of the B-chain. The isoelectric pH of the insulin is thereby moved from 5.4 towards physiological levels. Slightly acid solutions of derivatives, in which charge has been added in the C-terminus of the B-chain, have a prolonged action in vivo, in particular if the carboxyl group is blocked. It is found that the prolonged-acting hydrophilic insulins crystallize instantly when the pH is adjusted to 7. The prolonged action is ascribed to this readiness to crystallization combined with a low solubility, which may be further decreased by increased concentration of zinc ions. Hydrophobic insulins have a prolonged action independent of the site of substitution even if the derivative is soluble at physiological pH. Some derivatives were prepared from porcine insulin by tryptic transpeptidation. N-terminal B-chain substituted insulins were prepared by alkylation of a biosynthetic single-chain insulin precursor, followed by tryptic transpeptidation rendering the double chain insulin derivative. The observed blood glucose lowering in the rabbits implies that neither N- nor C-terminal B-chain substitution results in substantial deterioration of biological potency. An index for the degree of protraction based on the blood glucose data is used to compare the insulins.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.     

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.     

Study on preparation and unique properties of a novel insulin analogue with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain

A novel insulin analog, PIns, with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at human regular insulin B-chain was acquired through gene engineering. Preproinsulin for PIns was cloned and expressed using a bacterial expression system at a high level (72.1%) as fusion protein carrying a modified thioredoxin N-terminal region (1–21) linked to N-terminus of proinsulin by a lysine residue. Purified fusion protein was refolded and converted into PIns by a single enzymatic reaction. After PIns was purified, the homogeneity of it was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectronic focusing electrophoresis, amino acid composition analysis and mass spectrometry methods. A decreased tendency of self-association of PIns as compared with regular insulin was demonstrated by the size exclusion HPLC analysis. When subcutaneously administrated into normal rats, the PIns showed a faster rate of onset of action and a shorter duration of action compared with regular insulin, similar to the pharmacokinetic characteristics of insulin Lispro. These results showed that PIns is a rapid insulin analog. Furthermore, the N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain can be excised by DPPIV and recombinant peptidase with DPPIV-like activities. It is suggested that PIns serves as an artificial insulin precursor and can be transformed to regular insulin in vivo due to the truncation of N-terminal sequence of PIns B-chain by DPPIV.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.     

Sulfhydryl oxidation of reduced insulin in dilute solution

The reduction of insulin by tri-n-butylphosphine followed by air oxidation in dilute solution at pH 9.1 yields A- and B-chain disulfides. A(S-S)2 and B(S-S) have been purified on SP-Sephadex C-25 using a linear gradient of sodium chloride from 0.1 to 0.45 M in 0.5 M acetic acid containing 7 M urea. The overall yield of A(S-S)2 was 70%; and B(S-S), 60%. The A(S-S)2 and B(S-S) had the expected amino acid composition and N-terminal amino acid. The kinetics of reduction and reoxidation of insulin disulfide bonds are discussed.     

Degradation of proteins by enzymes exuded by Allium porrum roots–A potentially important strategy for acquiring organic nitrogen by plants

Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography–mass spectrometry (LC–MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.     

Purification,characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium,Halomonas sp. SK1

An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl-tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1T.     

Human gene 2 relaxin chain combination and folding

Relaxin is a small 6 kD two-chain peptide member of the insulin superfamily that is principally produced in the corpus luteum of the ovary and which plays a key role in connective tissue remodeling during parturition. Like insulin, it is produced on the ribosome as preprohormone that undergoes oxidative folding and subsequent proteolytic processing to yield the mature insulin-like peptide. In contrast to the now considerable insight into insulin chain folding and oxidation, comparatively little is known about the folding pathway of relaxin. A series of synthetic pairwise serine substituted relaxin A-chain cysteine analogues was prepared, and their oxidation behavior was studied both on their own and in the presence of native relaxin B-chain. It was observed that native S-reduced A-chain oxidized rapidly to a bicyclic product, whereas individual formation of each of the intramolecular disulfide bonds between Cys11 and Cys24 and the native Cys10 and Cys15 was considerably slower. Curiously, the non-native, isomeric Cys11-Cys15 disulfide bond formed most rapidly, although circular dichroism spectroscopy analysis showed this product to be devoid of secondary structure. This suggested that it may in fact be an intermediate in the subsequent formation of the native Cys10-Cys15 intramolecular disulfide. Combination of the native A-chain with the B-chain proceeded rapidly as compared with the A-chain analogue that lacked the intramolecular disulfide bond suggesting that this latter element is required as a first step in the folding process. It is therefore probable that relaxin is generated from its constituent A- and B-chains in a stepwise organization manner similar to that of insulin chain combination and folding. Further studies showed that the efficiency of combination of A-chain to B-chain was not markedly influenced by reaction temperature and that a reasonable yield of relaxin could be obtained on combination of the preoxidized A-chain with the S-reduced B-chain.     

Transamination of some sulphur- or selenium-containing amino acids by bovine liver glutamine transaminase.

S-(3-aminopropyl)cysteine and Se-(3-aminopropyl)selenocysteine are deaminated by bovine liver glutamine transaminase. The corresponding alpha-keto acids, S-(3-aminopropyl)-thiopyruvic acid and Se-(3-aminopropyl)selenopyruvic acid, are produced which spontaneously cyclize to ketimine derivatives. They have been identified by comparing their UV absorption spectra and some chemical or chromatographic properties with chemically synthesized authentic samples. Also S-(2-aminoethyl)homocysteine is the substrate for the enzyme. Kinetic parameters determined in comparison to thialysine and selenalysine show that neither the presence of a sulphur or a selenium atom nor the relative position of the atom in the carbon chain appreciably affects the substrate specificity of the enzyme. However, the length of the carbon chain has some influence on it.     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

CARBOXYMETHYLATION OF SULPHYDRYL GROUPS IN PROTEOLIPIDS1

(1) The sulphydryl groups of brain white matter proteolipids were studied by alkylation with iodoacetic acid and iodoacetamide in an organic solvent medium. To make sterically hindered sulphydryl groups available, the reaction was also carried out in the presence of sodium dodecyl sulphate. (2) In all cases, iodoacetamide was a better alkylating agent than was iodoacetic acid. (3) Only minimal alkylation of crude white matter proteolipids was obtained in the absence of detergent; addition of sodium dodecyl sulphate increased the availablity of SH groups. (4) Purified proteolipids prepared by column chromatography were alkylated to a lesser degree than were crude proteolipids. (5) Prior reduction with mercaptoethanol resulted in the quantitative conversion of cysteine to S-carboxymethylcysteine with either alkylating agent and in both preparations. (6) The possibility of a conformational difference between the protein in the crude and purified preparations is discussed.     

Degradation of L-djenkolate catalyzed by S-alkylcysteine alpha,beta-lyase from Pseudomonas putida

S-Alkylcysteine alpha, beta-lyase [EC 4.4.1.6] of Pseudomonas putida catalyzes alpha,beta-elimination of L-djenkolate [3,3'-methylenedithiobis(2-aminopropionic acid)] to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially. Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respectively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide. Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate. 1,2,4,5-Tetrathiane, 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and 1,2,3,5,6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the alpha,beta-elimination of L-djenkolate. In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate. When the alpha,beta-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed. Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.     

Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry.

The alkylation of cysteine residue by different classes of carbonium ions, derived from the cleavage of side chain protective groups in anhydrous HF, was investigated. It was found that side chain protection as beta-2,4-dimethylpent-3-yl ester (Dmp) or 2,4-dimethylpent-3-yloxycarbonyl (Doc) groups resulted in more than seven-fold lower level of alkylated byproducts. This makes Dmp and Doc protection of amino acid side chain during solid phase synthesis particularly valuable in the synthesis of peptides containing cysteine residues or other functional groups prone to alkylation by carbonium ions.     

Action of a cysteine proteinase from pupae of the blowfly Aldrichina grahami on the oxidized B-chain of bovine insulin

A cysteine proteinase purified from pupae of the blowfly (A. grahami) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Fifteen peptides were separated on HPLC using both gradient and isocratic elution methods. Analyses of amino acid content and N-terminal amino acids indicated that these were eleven homogeneous peptides produced by digestion and undigested insulin B-chain. Glu13-Ala14 and Tyr26-Thr27 were the major cleavage sites, and Asn3-Gln4, Cys7-Gly8, Tyr16-Leu17, Leu17-Val18 and Cys19-Gly20 were also often cleaved. These findings show the similarity between this enzyme and cathepsin L.     

Succination of proteins in diabetes

Cysteine is arguably the most reactive amino acid in protein. A wide range of cysteine derivatives is formed in vivo, resulting from oxidation, nitrosation, alkylation and acylation reactions. This review describes succination of proteins, an irreversible chemical modification of cysteine by the Krebs cycle intermediate, fumarate, yielding S-(2-succinyl)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane and develop in concert with mitochondrial and oxidative stress in diabetes. Increased succination of glyceraldehyde-3-phosphate dehydrogenase explains the loss in specific activity of this enzyme in muscle of streptozotocin-diabetic rats and increased succination of adiponectin may explain the decreased secretion of adiponectin from adipose tissue in type 2 diabetes. In addition to GAPDH and adiponectin, other succinated proteins identified in adipocytes include cytoskeletal proteins (tubulin, actin) and chaperone proteins in the endoplasmic reticulum. Succination of adipocyte protein in vitro is inhibited by uncouplers of oxidative phosphorylation and by inhibitors of ER stress. 2SC serves as a biomarker of mitochondrial stress and recent studies suggest that succination is the mechanistic link between mitochondrial and ER stress in diabetes.     

Study of the structural-energy aspects of complementary-addressed modifications of nucleic acids by reactive oligonucleotide derivatives having a 4-[N-methyl-N-(2-chloroethyl)amino[benzylidene group at the 3'end by a molecular mechanics method

Calculations of probabilities of the complementary addressed modification of a target nucleic acid by derivatives of oligonucleotides carrying a 4-[N-(2-chloroethyl)-N-methyl]aminobenzylidene group attached to the 3'-end (3'-BDO) have been made. The results show that the complementary complex of a target NA with 3'-BDO having R-configuration of carbon atom of the dioxalane ring is more stable than the complex including the S-stereoisomer. The S- and R-epimeres of 3'-BDO have essentially different positional abilities for alkylation of the target. The R-epimer alkylates best of all the third base of the target NA from terminal complementary pair of the complex. The S-epimer has another site the most sensitive to alkylation, which is a terminal complementary base of the target NA or the adjacent nonpaired base. Formation of the alkylation complexes are accompanied with a loss or a breakdown of hydrogen bonds in the terminal complementary base pair, thus decreasing the efficiency of alkylation. The modelling results are considered along with experimental data on modification which therefore can be interpreted on the fundamental structural level.